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D L Stout

Publications and source records attributed to D L Stout.

18 recordsLinked to original sources
PubMed1992

The role of transferrin in heme transport.

Porphyrin accumulation by proliferating cells, e.g., those associated with cancers or wounds, tends to correlate with increased transferrin receptor density. To determine whether transferrin might be implicated in porphyrin transport, fluorescence and absorption spectroscopy were used to study the interaction of porphyrins with transferrin. A single high-affinity binding site for heme and other porphyrins (Kd approximately 20-25 nM) was detected by fluorescence spectroscopy. Difference spectroscopy revealed three additional heme-binding sites. These sites were distinct from the iron-binding sites: 1) Apotransferrin and diferric transferrin bound porphyrins with equal affinity; 2) 59Fe was not displaced from transferrin by porphyrins. Murine erythroleukemia cells incubated with [59Fe]hemin-[125I]transferrin internalized both labels concomitantly. Accumulation of [59Fe]hemin could be blocked by a 100-fold excess of diferric transferrin but not by apotransferrin. These results indicate that cells can internalize exogenous heme, and possibly porphyrins, bound to transferrin via its receptor.

Animals↗
PubMed1990

Heme synthesis in normal mouse liver and mouse liver tumors.

Hepatic cancers from mice and rats demonstrate decreased levels of delta-aminolevulinic acid synthase, the rate-limiting enzyme in the heme synthetic pathway, and increased heme oxygenase, the heme-catabolizing enzyme. These findings suggest that diminution of P-450, b5, and catalase in these lesions may result from a heme supply that is limited by decreased heme synthesis and increased heme catabolism. Heme synthesis was measured in mouse liver tumors (MLT) and adjacent tumor-free lobes (BKG) by administering the radiolabeled heme precursors 55FeCl3 and [2-14C]glycine and subsequently extracting the heme for determination of specific activity. Despite reduced delta-aminolevulinic acid synthase activity in MLT, both tissues incorporated [2-14C]glycine into heme at similar rates. At early time points, heme extracted from MLT contained less 55Fe than that from BKG. This was attributed to the findings that MLT took up 55Fe at a slower rate than BKG and had larger iron stores than BKG. The amount of heme per milligram of protein was also similar in both tissues. These findings militate against the hypothesis that diminished hemoprotein levels in MLT result from limited availability of heme. It is probable, therefore, that decreased hemoprotein levels in hepatic tumors are linked to a general program of dedifferentiation associated with the cancer phenotype. Diminution of hemoprotein in MLT may result in a relatively increased intracellular heme pool. delta-Aminolevulinic acid synthase and heme oxygenase are, respectively, negatively and positively regulated by heme. Thus, their alteration in MLT may be due to the regulatory influences of the heme pool.

Animals↗
PubMed1987

Heme enzyme patterns in rat liver nodules and tumors.

Chemically induced rat hepatocyte nodules and carcinomas have a reduced capacity to oxidize drugs. The reduction in monoxygenase activity results largely from the partial loss of cytochrome P-450, a heme-containing terminal electron acceptor. To determine whether the cytochrome P-450 deficit was indicative of an altered heme metabolism, we quantitated four heme-containing proteins in normal rat liver and in rat liver nodules and cancers induced by 2-acetylaminofluorene or diethyl-nitrosamine: cytochrome P-450; cytochrome bs; catalase (EC 1.11.1.6); and tryptophan 2,3-dioxygenase (EC 1.13.11.11). The amounts of these components in nodules were 45%, 88%, 50%, and 59% of normal liver, respectively; in 2-acetylaminofluorene-induced cancers, 65%, 74%, 64%, and 65%, respectively; and in diethylnitrosamine-induced cancers, 40%, 69%, 56%, and 52%. delta-Aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in nodules were 95% and 138% of normal liver, respectively, whereas in 2-acetylaminofluorene-induced cancers, they were 47% and 233%, and in diethylnitrosamine-induced cancers, they were 50% and 175%. These data indicate that four nonmitochondrial liver hemoproteins were diminished to about the same extent in hepatic nodules and cancers. Nodules and cancers also demonstrated an increased capacity for heme degradation, while cancers also demonstrated a decreased capacity for heme synthesis. Thus, the resistance of nodules and tumors to P-450-activated cytotoxic agents may ultimately result from a disturbance in heme metabolism.

2-Acetylaminofluorene↗
PubMed1986

Protective role of thiols in carcinogen-induced DNA damage in rat liver.

Biological thiols are known to play an important role in the detoxification of xenobiotics, including chemical carcinogens. To determine the influence of cellular thiols on carcinogen induction of hepatic DNA damage in the rat, diethylmaleate (DEM) administration was used to deplete intracellular glutathione (GSH). The effects of administration of the synthetic thiols, N-acetylcysteine (NAC) and alpha-mercaptopropionylglycine (alpha MPG), on the induction of DNA lesions were also examined. Pretreatment with DEM reduced liver GSH levels by greater than 70%. As assessed by the technique of alkaline elution, subsequent administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in DNA damage 4 h post MNNG treatment which was 4- to 8-fold greater than that induced in the livers of rats treated with MNNG alone. However, DEM pretreatment had little effect on the extent of DNA damage induced by methylnitrosourea (MNU). DEM alone did not cause any measurable DNA damage. Pretreatment with alpha MPG or NAC reduced MNNG-induced DNA damage by as much as 77%. In contrast, MNU-induced DNA damage was increased by alpha MPG treatment whereas NAC treatment was without effect. These results indicated that in the rat liver, the activity of some DNA alkylating agents may be modulated in varying degree by the concentration of intracellular thiols. These data support the notion that thiols play an important role in protection against carcinogen damage, and that synthetic thiols such as alpha MPG and NAC may be useful as anti-carcinogenic agents against certain carcinogens.

Acetylcysteine↗
PubMed1986

Heme enzyme patterns in genetically and chemically induced mouse liver tumors.

Chemically induced rat hepatocyte nodules and hepatomas have repeatedly been shown to be deficient in Phase I drug-metabolizing enzymes. Some of these reduced activities are attributable to a diminution of the heme-containing terminal electron acceptor, cytochrome P-450. We recently demonstrated that spontaneous mouse liver tumors exhibit the same deficiency. Therefore, chemically induced and spontaneous liver tumors share common metabolic alterations which are likely to represent intrinsic characteristics of the tumorigenic process and are independent of its etiology. To determine whether the cytochrome P-450 deficit was the result of an altered heme metabolism, we quantitated four heme-containing proteins in normal mouse liver, spontaneous mouse liver tumors, and those induced by a single injection of diethylnitrosamine: cytochrome P-450; cytochrome b5; tryptophan 2,3-dioxygenase (EC 1.13.11.11); and catalase (EC 1.11.1.6). The amounts of these components in spontaneous tumors relative to normal liver were 0.35, 0.68, 0.76, and 0.51, respectively. Similar values were obtained with chemically induced tumors. The enzymes delta-aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in spontaneous tumor relative to liver were 0.49 and 1.51, respectively. Again, similar values were observed for the chemically induced tumors. Alteration of the latter two enzyme activities may be sufficient for the altered hemoprotein patterns seen in mouse liver tumors. Further, this pattern of metabolic alteration is common to both chemically induced and spontaneous tumors. Thus, tumor resistance to cytotoxic agents activated by the monooxygenase system is not necessarily induced by exposure to these agents, nor as a result of selection.

5-Aminolevulinate Synthetase↗
PubMed1986

Xenobiotic metabolizing enzymes in genetically and chemically initiated mouse liver tumors.

Chemically induced rat liver nodules and cancers characteristically demonstrate a limited capacity to activate xenobiotics to reactive species mainly because of decreased amounts of cytochrome P-450. These lesions also show enhancement of xenobiotic detoxication by such mechanisms as enzymic conjugation or reduction of cytotoxic species. We recently demonstrated a similar pattern of metabolic alteration in spontaneous mouse liver tumors. These findings suggested that certain phenotypic alterations attributed to chronic chemical exposure are inherent in the genetic program for carcinogenesis, and that they may arise independently of chronic exposure. To extend that study, we examined spontaneous and diethylnitrosamine-induced mouse liver tumors for nine enzyme activities commonly reported to be altered in chemically induced rat liver nodules and cancers. The activities of benzo(a)pyrene monooxygenase (EC 1.14.14.1), aminopyrene demethylase, cytochrome P-450 reductase, epoxide hydrolase (EC 3.3.2.3), and UDPglucuronosyl transferase (EC 2.4.1.17) in microsomes from spontaneous tumors relative to those from normal liver were 0.25, 0.43, 1.27, 0.90, and 0.51, respectively. Similar values were obtained with microsomes from chemically induced tumors. The activities of DT-diaphorase (EC 1.6.99.2), glutathione reductase (EC 1.6.4.2), glutathione S-transferase (EC 2.5.1.18), and glutathione peroxidase (EC 1.11.1.9) in cytosol from spontaneous tumors relative to cytosol from normal liver were 2.24, 2.0, 2.43, and 0.31, respectively. Similar values were obtained with cytosol from chemically induced tumors. These results demonstrated that a significant portion of the enzymic phenotype observed in chemically induced rat liver nodules and cancers, which may confer resistance to cytotoxic chemicals, is manifest in spontaneous and chemically induced mouse liver tumors. Further, initiated cells that exhibit this phenotype replicated and progressed in the absence of continued chemical selection.

Aminopyrine N-Demethylase↗
PubMed1984

A constitutive deficiency in the monooxygenase system of spontaneous mouse liver tumors.

Exposure to chemical carcinogens evokes a population of altered hepatocytes that demonstrates significantly diminished monooxygenase activity. It has been suggested that this alteration permits the target cell to escape the toxic effects of the carcinogen and proliferate. In an attempt to determine whether this enzyme defect has broader implications for the carcinogenic process, we examined the monooxygenase system and additional components of spontaneous hepatocellular tumors in mice with a genetic predisposition to tumorigenesis. These tumors uniformly demonstrated a significant deficit in cytochrome P-450 and aminopyrine N-demethylase, despite the absence of known carcinogens, toxins, or promoting agents in their environment. Tumors of similar histiotype induced by a small, single neonatal administration of diethylnitrosamine demonstrated identical alterations. This report, therefore, suggests a strong link between a genetic program for tumorigenesis and a deficit in the monooxygenase system in spontaneous tumors. Further, it reveals that a toxic-selective environment is not required for the expansion of the cell population that possesses this phenotype.

Animals↗
PubMed1982

Occurrence of progressive DNA damage coincident with the appearance of foci of altered hepatocytes.

The technique of alkaline elution was used to evaluate alterations in nuclear DNA obtained from livers of rats that had received a single 6.6 mg/kg dose of diethylnitrosamine (DEN) during liver regeneration and subsequent exposure (7 days after partial hepatectomy) to phenobarbital (BP). DNA from normal and regenerating liver demonstrated a significant increase in the rate of elution following DEN administration. In those DEN-treated groups that did not receive PB, the rate of DNA elution decreased slowly but failed to return to normal by 44 weeks. Exposure to PB hastened recovery of a normal DNA elution profile in normal liver following DEN treatment, by 44 weeks, DNA from these rats eluted at a normal rate. However, in rats treated with DEN during liver regeneration, the rate of DNA elution began to increase at 28 weeks of PB exposure and became progressively more rapid through the 36th and 44th weeks. This latter group also demonstrated foci of gamma-glutamyl-transferase (GGT)-positive hepatocytes at 28 weeks of PB exposure that increased in number and size concomitantly with the increasing rate of DNA elution. AT 44 weeks, one or more primary hepatocellular carcinomas were present in 73% of the rats in this group; none was seen in any other group. Foci of GGT positive hepatocytes, an increasing rate of DNA elution and eventual primary hepatocellular carcinoma were seen in a group of rats that was begun on PB as late as 85 days after partial hepatectomy and DEN treatment.

Animals↗
PubMed1980

Progressive DNA damage in hepatic nodules during 2-acetylaminofluorene carcinogenesis.

The method of alkaline elution was used to detect DNA alteration in rat liver throughout the carcinogenic sequence that resulted from exposure to a standard four-cycle feeding regimen of 2-acetylaminofluorene (AFF). At the end of 3 weeks of AAF feeding, DNA from aliquots of whole liver demonstrated a small but significant degree of damage. By the end of the fourth AAF feeding, the liver exhibited numerous nodules that could be dissected free from surrounding tissue. DNA from these putative premalignant lesions showed approximately 136% more damage than that seen at the end of 3 weeks of AAF feeding. Two to 4 months following the cessation of AAF, DNA from persistent nodules was examined for eivdence of alteration. Despite the prolonged absence of exposure to AAF, DNA damage was found to have progressed and was as much as 320% greater than that seen at the end of the first cycle. While the persistence of DNA damage during chronic exposure to a carcinogen has been reported previously, the observations that the DNA of a focal lesion which is putatively premalignant was damaged and, further, that this alteration progressed in the absence of a carcinogen, are unique.

2-Acetylaminofluorene↗
PubMed1980

Covalent binding of 2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-acetylaminofluorene to rat liver nuclear DNA and protein in vivo and in vitro.

Binding of the hepatocarcinogen 2-acetylaminofluorene (AAF) and two metabolites, 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF), to the DNA and protein of rat hepatic nuclei was examined in vitro and in a cell-free system. Three and one-half hr following a single injection of each compound in equimolar amounts. DNA contained approximately 50% more of the compounds per mg than did protein. The amount of N-OH-AAF bound to DNA was 4 times greater than that of AAF, while AF bound in intermediate amounts. When incubated with nuclei in a cell-free system, AAF seldom bound in measurable amounts, while significant amounts of N-OH-AAF and AF bound to both DNA and protein. As occurred in vivo, DNA bound more of each per mg than did protein. The amount of N-OH-AFF bound to intranuclear DNA increased 54% when an aliquot of the postmicrosomal liver fraction was added to the incubation mixture, but maximum binding of AF occurred in the absence of any other liver fraction. Thus, it was shown that two AAF metabolites, AF and N-OH-AAF, bind covalently to the DNA and protein of hepatic nuclei more readily than does AAF itself and that binding in a cell-free system parallels binding in vivo. Additional evidence suggests that rat hepatic nuclei are capable of mediating the binding of AF and N-OH-AAF to macromolecules through distinct enzyme systems. This is the first demonstration that the nucleus is capable of metabolizing AF to an electrophile that can bind covalently to DNA.

2-Acetylaminofluorene↗
PubMed1976

N'-hydroxy-2-aminofluorene: the principal mutagen produced from N-hydroxy-2-acetylaminofluorene by a mammalian supernatant enzyme preparation.

A Salmonella typhimurium TA 1538 culture system wasused to monitor the production of mutagen from N-hydroxy-2-acetylaminofluorene by soluble liver enzymes. When benzene was used to extract the mutagen, no mutagenic activity remained in the liver enzyme preparation. The benzene extract contained approximately two-thirds of the total mutagenic activity produced by the liver enzyme preparation. Using thin layer and column chromatography to analyze the benzene extract, we deduced that N-hydroxy-2-aminofluorene accounted for the mutagenic activity which resulted from the incubation of N-hydroxy-2-acetylaminofluorene with soluble liver enzymes.

2-Acetylaminofluorene↗
PubMedDate not supplied

The effects of tin-protoporphyrin administration on hepatic xenobiotic metabolizing enzymes in the juvenile rat.

The heme analogue tin-protoporphyrin IX (SnP) is a potent inhibitor of microsomal heme oxygenase. Administration of SnP to neonatal rats can prevent hyperbilirubinemia by blocking the postnatal increase of heme oxygenase activity. Apparently innocuous at therapeutic doses, it is of potential clinical value for chemoprevention of neonatal jaundice. We found that when 50-g male Sprague-Dawley rats were treated daily with 50 mumol of SnP/kg sc for 6 days, hepatic microsomal cytochromes b5 and P-450 were significantly diminished. Cytochrome P-450 reductase, two P-450-dependent monooxygenases, aminopyrine demethylase and benzo(a)pyrene hydroxylase, and catalase, a peroxisomal hemoprotein, were also significantly diminished. These results suggested that SnP might significantly affect the metabolism of other xenobiotics. This possibility was confirmed by the finding that hexobarbital-induced sleep lasted 4 times longer in SnP-treated rats than in controls. Inhibition of protein synthesis by SnP was ruled out as the cause of hemoprotein loss when administration of [3H]leucine to SnP-treated and control rats demonstrated that proteins of the microsomal, cytosolic, and plasma membrane fractions of the livers from both groups incorporated similar levels of leucine. When 55FeCl3 and [2-14C]glycine were administered to measure heme synthesis, heme extract from the livers of SnP-treated rats contained 4 times more label from iron and glycine than did heme from control livers. Despite the apparent increased rate of heme synthesis in SnP-treated rats, each of the three cell fractions demonstrated a significant loss of heme but contained sizable amounts of SnP. These findings suggest that SnP causes a decrease of functional hemoprotein and partial loss of enzymic activity by displacing intracellular heme.

Animals↗